by Po Chu, Leung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-169). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abbreviation --- p.iii / Abstract --- p.vii / Chinese Abstract --- p.x / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- The Development of Hematopoietic Progenitor Cells --- p.1 / Chapter 1.1.2 --- Hematopoietic Growth Factors --- p.3 / Chapter 1.1.3 --- Transcriptionl Factors Involved in Lineage Commitment of Hematopoietic Progenitor Cells --- p.5 / Chapter 1.2 --- Leukemia --- p.7 / Chapter 1.2.1 --- Occurrence and Classification of Leukemia --- p.7 / Chapter 1.2.2 --- The Pathological Features and Etiology of Leukemia --- p.10 / Chapter 1.2.3 --- The Molecular Basis of Leukemia --- p.13 / Chapter 1.2.4 --- Current Therapeutic Strategies --- p.14 / Chapter 1.2.4.1 --- Conventional Therapies for Leukemia --- p.14 / Chapter 1.2.4.2 --- Induction of Cell Differentiation and Apoptosis for Treatment of Leukemia --- p.16 / Chapter 1.2.5 --- The Use of Murine Myelomonocytic Leukemia WEHI-3B JCS Cells As a Model for the Study of Leukemia Cell Proliferation， Differentiation and Survival --- p.22 / Chapter 1.3 --- Taxol: A Novel Anti-cancer Agent --- p.23 / Chapter 1.3.1 --- Discovery and Action Mechanism --- p.23 / Chapter 1.3.2 --- Metabolism and Toxicity of Taxol --- p.27 / Chapter 1.3.3 --- The Biological Activities of Taxol --- p.28 / Chapter 1.3.4 --- The Anti-tumor Effects of Taxol --- p.30 / Chapter 1.3.5 --- The Effects of Taxol on Leukemia --- p.31 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.32 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Mice --- p.35 / Chapter 2.1.3 --- "Culture Media,Buffer and Other Solutions" --- p.37 / Chapter 2.1.4 --- Radioisotope and Scintillation Fluid --- p.39 / Chapter 2.1.5 --- Taxol --- p.40 / Chapter 2.1.6 --- Recombinant Cytokines --- p.40 / Chapter 2.1.7 --- Vitamin Analogs --- p.42 / Chapter 2.1.8 --- Various Signal Transduction Pathway Activators and Inhibitors --- p.42 / Chapter 2.1.9 --- Monoclonal Antibodies and Buffers for Flow Cytometry --- p.43 / Chapter 2.1.10 --- Reagents and Chemicals for Gene Expression Study --- p.45 / Chapter 2.1.11 --- Chemical Solutions and Buffers for Western Blot --- p.50 / Chapter 2.1.12 --- Reagents for Colony Assay --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Culture of Leukemia Cell Lines --- p.55 / Chapter 2.2.2 --- Treatment of Leukemia Cells with Various Drugs and Cytokines --- p.55 / Chapter 2.2.3 --- Cell Morphological Study --- p.55 / Chapter 2.2.4 --- Determination of Leukemia Cell Survival and Proliferation --- p.56 / Chapter 2.2.5 --- Colony Assay --- p.56 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.57 / Chapter 2.2.6.1 --- Surface Antigen Immunophenotyping --- p.57 / Chapter 2.2.6.2 --- Assay of Endocytic Activity --- p.58 / Chapter 2.2.6.3 --- Cell Cycle /DNA Content Evaluation --- p.58 / Chapter 2.2.7 --- Gene Expression Study --- p.59 / Chapter 2.2.7.1 --- Preparation of Total Cellular RNA --- p.59 / Chapter 2.2.7.2 --- Reverse Transcription --- p.60 / Chapter 2.2.7.3 --- Polymerase Chain Reaction (PCR) --- p.60 / Chapter 2.2.7.4 --- Agarose Gel Electrophoresis --- p.61 / Chapter 2.2.8 --- DNA Fragmentation Analysis --- p.61 / Chapter 2.2.9 --- Protein Expression Study --- p.62 / Chapter 2.2.9.1 --- Protein Extraction --- p.62 / Chapter 2.2.9.2 --- Quantification of the Protein --- p.62 / Chapter 2.2.9.3 --- Western Blot Analysis --- p.63 / Chapter 2.2.10 --- Statistical Analysis --- p.64 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Effects of Taxol on the Proliferation and Apoptosis of the Murine Myeloid Leukemia Cells --- p.65 / Chapter 3.1.1 --- Growth-Inhibitory Effects of Taxol on Murine Myeloid Leukemia WEHI-3B JCS cells --- p.65 / Chapter 3.1.2 --- Cytotoxic Effects of Taxol on Murine Bone Marrow Cells and Myeloid Leukemia WEHI-3B JCS Cells --- p.69 / Chapter 3.1.3 --- Anti-proliferative Effect of Taxol on Different Leukemia Cell Lines --- p.72 / Chapter 3.1.4 --- Effects of Taxol on the Cell Cycle Kinetics of WEHI-3B JCS Cells --- p.81 / Chapter 3.1.5 --- Induction of DNA Fragmentation of WEHI-3B JCS cells by Taxol --- p.83 / Chapter 3.1.6 --- Effect of Taxol on the Clonogenicity of WEHI-3B JCS Cells In Vitro and Tumorigenicity In Vivo --- p.86 / Chapter 3.2 --- Effects of Taxol on the Induction of Monocytic Cell Differentiation in Murine Myeloid Leukemia Cells --- p.88 / Chapter 3.2.1 --- Morphological Changes in Taxol-Treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.88 / Chapter 3.2.2 --- Surface Antigen Immunophenotyping of Taxol-treated WE HI-3B cells --- p.91 / Chapter 3.2.3 --- Endocytic Activity of Taxol-treated WEHI-3B JCS cells --- p.95 / Chapter 3.3 --- Modulatory Effect of Taxol and Cytokines on the Proliferation of WEHI- 3B JCS Cells --- p.96 / Chapter 3.4 --- Modulatory Effect of Taxol and Physiological Differentiation Inducers on the Proliferation of WEHI-3B JCS cells --- p.103 / Chapter 3.5 --- The Possible Involvement of Protein Kinase C in the Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.106 / Chapter 3.6 --- Modulation of Apoptotic Gene Expression in Taxol-treated WEHI-3B JCS cells --- p.113 / Chapter 3.7 --- Modulatory Effects of Taxol on the Protein Expression of WEHI-3B JCS Cells --- p.119 / Chapter Chapter 4: --- Discussion and Conclusions / Chapter 4.1 --- "Effects of Taxol on the Proliferation,Differentiation and Apoptosis of the Murine Myeloid Leukemia Cells" --- p.126 / Chapter 4.2 --- "Modulatory Effects of Taxol, Cytokines and Physiological Differentiation Inducers on the Proliferation of the Myelomonocytic Leukemia WEHI-3B JCS Cells" --- p.132 / Chapter 4.3 --- The Possible Involvement of Protein Kinase C in Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.136 / Chapter 4.4 --- The Modulation of Apoptosis Gene Expression in Taxol-treated WEHI-3B JCS Cells --- p.137 / Chapter 4.5 --- The Modulation of Protein Expression in Taxol-treated WEHI-3B JCS Cells --- p.138 / Chapter 4.6 --- Conclusions and Future Perspectives --- p.139 / References --- p.141

by Li Jian Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 157-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Colon cancer is one of the most common cancers in both the United States (US) and throughout the world. Over the last 30 years, despite the development of multiple classes of effective anti-tumor agents, colon cancer has consistently remained the second leading cause of mortality amongst all cancers and is today responsible for over 50,000 deaths a year in the US alone. Among the greatest challenges to the successful treatment of colon cancer is its heterogeneity in terms of drug-sensitivity, whereby it is often difficult to identify which patients will benefit from a specific class of anti-tumor agents before treatment has begun. It is therefore imperative to identify predictive biomarkers that can be leveraged to distinguish which colon tumors are most likely to respond to individual anti-cancer drugs. This will help develop new therapeutic algorithms that can maximize patient survival by rapidly matching individual patients with the specific treatment combinations that are most likely to benefit them as well as sparing them the toxicities from drugs that would be ineffective. Previous studies have reported that human colon carcinomas lacking expression of the caudal-type homeobox 2 (CDX2) transcription factor can be leveraged as a predictor of benefit from adjuvant chemotherapy containing 5-fluorouracil (5-FU). Lack of CDX2 expression associates with microsatellite instability (MSI), as well as several histopathological and molecular features that associate with exceptionally poor prognosis such as poor differentiation, lympho-vascular invasion, and BRAF mutation. However, the molecular mechanisms linking lack of CDX2 expression with increased drug sensitivity are currently unknown. In the first section of this study, we conducted a high throughput screen (HTS) aimed at identifying clinically approved anti-tumor drugs that display selective activity against colon carcinomas lacking CDX2 expression (CDX2-negative). The results of our screening, which compared an isogenic pair of CDX2+/+ and CDX2-/- cell lines generated by genetic inactivation of CDX2 using CRISPR/Cas9 constructs, revealed that CDX2-negative colon cancer cells display increased sensitivity to anti-tumor drugs that are substrates of the ATP binding cassette sub-family B member 1 (ABCB1) transporter. ABCB1 is a drug-efflux protein known for its capacity to extrude multiple classes of anti-tumor agents from the cytoplasm, therefore contributing to drug-resistance in cancer cells. Importantly, analysis of CDX2 and ABCB1 expression in two independent gene-expression databases (NCBI-GEO: n=2115; TCGA: n=478) revealed that a lack of CDX2 expression is invariably associated with lack of ABCB1 expression in human primary colon carcinomas. Furthermore, our molecular studies revealed that forced expression of CDX2 in human CDX2-negative colon cancer cells was capable of inducing expression of ABCB1, while genetic inactivation of CDX2 in human CDX2-positive cancer cells using CRISPR/Cas9 constructs resulted in loss of ABCB1 expression, thus establishing CDX2 as a direct mechanistic regulator of ABCB1 expression. Amongst all of the anti-tumor drugs identified as being ABCB1 substrates with preferential activity against CDX2-negative colon cancer cells, we observed that paclitaxel was the FDA-approved drug with the greatest degree of selectivity with a 10-fold difference in IC50. When tested in vivo against a collection of human patient derived xenograft (PDX) lines representative of both CDX2-negative and CDX2-positive colon carcinomas, paclitaxel displayed selective activity against CDX2-negative models, often inducing volumetric regression of established lesions. Our study, therefore, identified paclitaxel as a clinically approved anti-tumor agent that should be investigated for use in the treatment of CDX2-negative colon carcinomas. In the second portion of our study, we sought to conduct a preliminary evaluation of the possibility of using immune checkpoint inhibitors (ICIs) for the treatment of CDX2-negative colon carcinomas. ICIs have been shown to display substantial anti-tumor activity against colon carcinomas with microsatellite instability (MSI) and against epithelial malignancies over-expressing the immune-suppressive molecule PD-L1/CD274. Because CDX2-negative tumors are enriched for MSI and high levels of PD-L1/CD274, they are predicted to include a subgroup that is responsive to ICIs. However, not all MSI tumors respond to ICIs and, contrary to the majority of MSI tumors, the subgroup of MSI tumors characterized by a CDX2-negative phenotype is often associated with poor prognosis. Because the clinical activity of ICIs is dependent upon expression of class-I HLA molecules by tumor cells, we decided to evaluate whether CDX2-negative tumors were associated with inactivating mutations in class-I HLA genes. Our attention focused on a highly conserved poly-cytosine repeat region in the coding sequence of HLA-A (c.621_627) and HLA-B (c.621_626) genes. Because this sequence fulfilled the molecular definition of microsatellite, we predicted it to be highly susceptible to frameshift mutations (insertions or deletions) in MSI colon tumors. Indeed, a search across three independent genetic databases (TCGA, COSMIC, EBI) confirmed that this highly conserved poly-cytosine repeat region was targeted by recurrent and deleterious mutations in at least one HLA-A or HLA-B allele of at least 13% (n=21/156) of human MSI colon tumors, as compared to 0.3% (n=2/770) of human colon tumors with a microsatellite stable (MSS) phenotype (p<0.0001). Among tumors assessable for CDX2 expression, this specific type of class-I HLA mutations was more frequent among CDX2-negative (12%; n=6/49) as compared to CDX2-positive (1.5%; n=5/340) colon tumors (p<0.001), but was similar within MSI CDX2-negative (21%; n=6/28) and MSI CDX2-positive (17%; n=5/30) subgroups. In summary, this work achieved two main results: 1) it identified paclitaxel, a clinically approved anti-tumor drug, as a new treatment option for patients with CDX2-negative colon cancers, which represents an extremely aggressive subgroup of colorectal malignancies; 2) it revealed that, in human MSI colon tumors, class-I HLA genes are prone to recurrent frameshift mutations in a genomic hotspot, mutations that are likely to associate with tumor resistance to ICIs and that they are therefore likely to represent a new class of actionable predictive biomarkers for both MSI and CDX2-negative colon carcinomas. These findings will help advance our understanding of colon cancer biology, and hopefully improve treatment algorithms for the clinical management of colon cancer patients.

Chemotherapy persists as one of the mainstays of breast cancer treatment, particularly for triple-negative breast cancer which currently has no targeted treatment methods. While chemotherapy is beneficial for killing the malignant tumor cells, it leads to the release of damage-associated molecular patterns into the tumor microenvironment. Damage-associated molecular patterns are a contributing factor to cancer-related inflammation which can potentiate metastatic spread through several mechanisms such as the development of tumor microenvironments at metastastic sites. These damage-associated molecular patterns include nucleic acids, nucleic acid-associated lipids and vesicles, cytokines, and proteins such as high mobility group protein B1. Polyamidoamine (PAMAM) is a biodegradable, water-soluble dendrimer polymer with the ability to possess different charges and sizes depending on its terminal branches and degree of branching (i.e. generation number), respectively. Amine-terminated PAMAM-NH2 is positively charged and can bind to circulating DNA and RNA. Since most DAMP molecules are negatively charged, I hypothesized that a polycation such as PAMAM-NH2 would be an efficient nanomaterial to remove pathogenic NA DAMPs generated by chemotherapy. Building on this dendrimer, we synthesized modified cationic PAMAM-generation 3 derivatives with an aim to balance toxicity with NA-binding affinity and capacity to encapsulate chemodrugs. Our results found that these soluble and nanoparticle PAMAM materials can bind to both cell-free DNA and RNA released as a result of treating triple-negative breast cancer cells with chemotherapy drugs such as doxorubicin and paclitaxel. These PAMAM-G3 materials are termed as nucleic acid binding polymers and nucleic-acid binding polymeric nanoparticles.My thesis dissertation explores the anti-metastatic effects of nucleic-acid binding polymeric nanoparticles delivering the chemotherapy drug paclitaxel using in-vitro and in-vivo models. Two murine metastatic breast cancer models served as the basis for assessing the effects of conventional paclitaxel delivery compared to paclitaxel delivery from within PAMAM nucleic-acid binding polymeric nanoparticles with respect to primary tumor growth, extent of lung metastasis, and the systemic inflammatory response reflected in murine serum. Compared to treatment with unencapsulated paclitaxel, delivery of paclitaxel within the PAMAM nucleic-acid binding polymeric nanoparticles resulted in significantly decreased serum cell-free DNA levels, decreased inflammatory cytokines, and a lower degree of lung metastasis in the mice. The decrease in the degree of lung metastasis in mice receiving paclitaxel within the PAMAM nanoparticles was confirmed by assessing the photon flux signal of 4T1-luciferase breast cancer cells invading the murine lungs in both in-vivo and ex-vivo imaging and by using a machine learning method to quantify the degree of metastasis in H&E- stained sections of the lungs. The ability to mitigate the phenomenon of chemotherapy-induced cancer metastasis while effectively delivering the chemotherapy to the tumor microenvironment could help improve the outcomes of patients being treated with chemotherapy. This work developed a therapeutic cationic PAMAM nanocarrier-based strategy to inhibit paclitaxel-induced metastasis by scavenging cell-free nucleic acids and mitigating cell-free nucleic acid-induced inflammation.

Indiana University-Purdue University Indianapolis (IUPUI) / Resveratrol has been shown to inhibit cell growth and induce apoptosis, as well as augment chemotherapeutics and irradiation in multiple cancer types. However, it is unknown if resveratrol is beneficial for treating drug-resistant cancer cells. To study the effects of resveratrol in triple negative breast cancer cells that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the MDA-MB-231 breast cancer cell line. The resulting cell line, MDA-MB-231/PacR, exhibited a 12-fold increased resistance to paclitaxel but remained sensitive to resveratrol treatment. Resveratrol treatment reduced cell proliferation and colony formation and increased senescence and apoptosis in both the parental MDA-MB-231 and MDA-MB-231/PacR cell lines. Importantly, resveratrol treatment augments the effects of paclitaxel in both cell lines. The expression of the drug efflux transporter gene, MDR1, and the main metabolizing enzyme of paclitaxel gene, CYP2C8, was increased in the resistant cells. Moreover, pharmacological inhibition of the protein products of these genes, P-glycoprotein and CYP2C8, decreased paclitaxel resistance in the resistant but not in the parental cells, which suggests that the increase of these proteins are important contributors to the resistance of these cells. In conclusion, these studies imply that resveratrol, both alone and in combination with paclitaxel, may be useful in the treatment of paclitaxel-sensitive and paclitaxel-resistant triple negative breast cancers.